Cytochrome P450 3

The metalloenzymes that make up the group cytochrome P-450, or CYP, play an integral and varied role in biological systems. Originally discovered in rat liver tissue by researchers in 1955, CYP has since been found in other tissues of animals, plants, fungi, and even bacteria (Lewis 1996). Understanding the functions of CYP in these biological systems unequivocally requires an understanding of CYP’s basic molecular structure and the reactivity that arises from its molecular properties.

Molecular Structure

CYP enzymes belong to a group of proteins that contain a prosthetic iron-heme cofactor. Accordingly, CYP enzymes are termed ‘hemoproteins.’ Iron holds two 4s electrons and six 3d shell electrons in its valence shell. The metalloenzyme’s reactivity depends on the shifting oxidation state of iron between ferrous Fe²⁺ (loss of the 4s electrons) and ferric Fe³⁺ (loss of an additional 3d electron) (Lewis 1996). Because the ferric state results in a half-filled 3d shell, it is the more stable form of the two states. As a result, CYP can be readily reduced with the addition of an electron.

The iron center in CYP is bonded to a protoporphyrin IX macromolecule and two axial ligands. The first axial ligand is a thiolate group that is attached to the adjunct protein. The second axial ligand varies depending on the enzymatic cycle (Shaik 2005). However, in CYP’s original state, water generally occupies the axial position. Figure 1 illustrates the metal complex structure of CYP. Figure 2 shows the complete structure of a specific CYP enzyme, cytochrome P-450 Oxidase (CYP2C9).

Figure 1. The metal complex of cytochrome P-450 (Shaik 2005)

Figure 2. The structure of cytochrome P-450 Oxidase (CYP2C9) (Wikipedia.org)

The iron center and the atoms immediately bonded to the center have octahedral geometry. The iron complex has a C₄ principle axis but no perpendicular C₄ axes. Because the center has 4 σ_v planes that contain the principle C₄ axis, the metal complex belongs to the C_4v point group. The complete CYP enzyme, which contains the adjunct protein, lacks symmetry and therefore belongs to the C₁ point group.

Vibrational Spectroscopy

Both Resonance Raman (RR) and Infrared (IR) spectroscopy have been used to study the structure of CYP. RR spectroscopy is particularly useful for identifying the structures of different redox and ligation states (Parthasarathi, et.al., 1987). As a result, RR has been employed to study vibrational transitions associated with the metal-ligand bonds in CYP and has provided useful insight regarding the varying oxidation and ligation states of the active site. Table 1 lists some of the distinguishing differences in the RR spectra of CYP in the Fe²⁺ and Fe³⁺ oxidation states.

		Frequency Range (cm^-1)
Mode	Symmetry	Fe(III)	Fe(II)
v10	B1g	1623-1637	1600-1612
v37	Eu	1580-1601	1584-1586
v19	A2g	1583	--
v2	A1g	1565-1584	1556-1564
v11	B1g	1549-1564	1532-1534
v38	Eu	1548-1550	1521
v3	A1g	1485-1502	1462-1466
v28	B2g	1464-1465	1445
v4	A1g	1370-1373	1341-1344

Table 1. Differences in the RR Spectra of CYP in the Fe²⁺ and Fe³⁺oxidation states (Lewis 1996)

IR spectroscopy has also been employed to investigate the effect of changes in the heme environment on heme-ligand stretching (Lewis 1996). Using the equation 3N-6 for non-linear molecules where N is the number of atoms bonded to the iron center, it can predicted that the 6-coordinated iron complex in CYP would generate 12 total stretching vibrational modes.

Catalytic Cycle

CYP can catalyze a number of reactions, including various oxidation reactions. In 1968, researchers proposed an enzymatic cycle that is commonly accepted today as the general process for CYP mono-oxygenation (Denisov, et. at., 2005). In this cycle, a substrate binds to the low-spin ferric enzyme, effectively displacing the water ligand that is coordinated to the central heme and changing the complex to a high spin-state. As a result of this shift from a low spin to a high spin state, the complex has a greater reduction potential and is more easily reduced than the original ligated heme. An oxygen molecule then binds to the heme center, forming an oxy-heme compex. The reduction of this complex, followed by two subsequent protonations and heterolysis of the O-O bond results in the formation of the original heme enzyme ligated with water and an oxygenated substrate product. Figure 3 summarized the CYP enzymatic mono-oxygenation catalytic cycle.

Figure 3: The Cytochrome P-450 Catalytic Cycle (Wikipedia.org)

The monoxygenase (hydroxylation) reaction can also represented by Equation 1 (Sato, et. al., 1978).

Eq 1. RH + 2H⁺ + O₂ + 2e^{- -->} ROH + H₂O

In this reaction, RH denotes the substrate and ROH denotes the final hydroxylated metabolite. Hydroxylation transforms a nonpolar xenobiotic to a polar product by adding a polar handle (-OH) to the molecule.

Biological Role

CYP has two primary functional roles in the human body. First, CYP is used by the body to metabolize and transform a range of hydrophobic xenobiotics (exogenous, foreign compounds such as pesticides, carcinogens, and pollutants) to more polar metabolites so that they can be more readily excreted in the urine (Denisov, 2005). In this respect, by biotransforming potentially toxic compounds to less potent forms, CYP works as a natural detoxifying agent of the body. Not surprisingly, large populations of CYP are found bound to the endoplasmic reticulum in mammalian liver cells, the primary site of metabolism. The gene families most commonly found in humans and involved in metabolism include cytochrome P-450 1, 2, and 3 (known as CYP 1, CYP 2, and CYP 3) (Sato 1978).

Second, CYP enzymes are used in the synthesis of important signaling molecules, such as steroid hormones in the endocrine glands and fat-soluble vitamins (Sato 1978). CYP metabolism can have adverse effects as well beneficial effects. Rather than deactivating a xenobiotic and facilitating its clearance from the body, CYP can also do just the opposite. CYP can bioactivate drugs and other chemicals, transforming the xenobiotics to products that are more reactive—and more damaging—than their original forms (Guengerich 2006). For instance, it is believed that CYP plays an important role in activating carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), by an oxidation mechanism (Degawa, et. al. 1994). Drugs are also susceptible to detrimental bioactivation (Guengerich 2006). As a result, predicting drug reactivity with CYP and drug toxicity is a critical step in the development of pharmaceuticals.

Conclusion

Biological utilization of CYP is intimately tied to CYP structure and reactivity.

References

1. IMG: Cytochrome P-450 Oxidase (CYP2C9). http://en.wikipedia.org/wiki/File:Cy...idase-1OG2.png
2. Degawa, M., S. Stern, M. Martin, F. Guengerich, P. Fu, K. Ilett, R. Kaderlik, F. Kadlubar. Metabolic Activation and Carcinogen-DNA Adduct Detection in Human Larynx. Cancer Research, 1994, 54, 4915-4919.
3. Denisov, I.G., T. M. Makris, S. G. Sligar, I. Schlichting. Structure and Chemistry of Cytochrome P450. Chem. Rev., 2005, 105, 2253-2277
4. Guengerich, F.P. Cytochrome P450s and Other Enzymes in Drug Metabolism and Toxicity. AAPS, 2006, 8 (1), 101-111.
5. Lewis, D. F. V., 1996. Cytochromes P450: Structure, Function and Mechanism. Bristol: Taylor & Francis.
6. Parthasarathi, N., C. Hansen, S. Yamaguchi, T. G. Spiro. Metalloporphyrin core size resonance Raman marker bands revisited: implications for the interpretation of hemoglobin photoproduct Raman frequencies. J. Am. Chem. Soc., 1987, 109 (13), 3865–3871.
7. Sato, R. and T. Omura. , 1978. Cytochrome P-450. New York: Academic Press.
8. Shaik, S., S. P. de Visser, 2005. Cytochrome P450: Structure, Mechanism, and Biochemistry. Ortiz de Montellano, P. (ed.), New York: Kluwer Academic.
9. IMG: The P450 catalytic cycle. Wikipedia.org. <http://en.wikipedia.org/wiki/File:P450cycle.svg>